RS59210B1 - Stem cell therapy in endometrial pathologies - Google Patents

Description

Description

FIELD OF INVENTION

[0001] The present invention generally relates to the use of autologous CD133+ bone marrow stem cells (BMDSC) to initiate endometrial regeneration and treat endometrial pathological conditions such as Asherman's syndrome and endometrial atrophy.

BACKGROUND OF THE INVENTION

[0002] In women of reproductive age, two layers of the endometrium can be distinguished: (i) the functional layer next to the uterine cavity, and (ii) the basal layer, which is next to the myometrium and below the functional layer. The functional layer is formed after the end of menstruation during the first part of the previous menstrual cycle. Proliferation is induced by estrogen (follicular phase of the menstrual cycle), and later changes in this layer produce progesterone from the corpus luteum (luteal phase). It is adapted to provide an optimal environment for the implantation and growth of the embryo. This layer is completely emptied during menstruation. In contrast, the basal layer is not shed at any time during the menstrual cycle. The regeneration of the human endometrium under the systemic changes of ovarian steroids in each menstrual cycle is essential to prepare this organ for its main function, ie, the development of the endometrial implantation window for the placement of the implanting blastocyst, allowing pregnancy to occur. Therefore, renewal of all cellular parts of the functional endometrial layer with each menstrual cycle is necessary for normal reproductive function.

[0003] Asherman's syndrome (AS) is a condition in which there is destruction of the endometrium caused by repeated or aggressive curettages and/or endometritis. It creates obliteration of the uterine cavity with intrauterine adhesions and absence of functional endometrium in many areas. Women with this disease as well as with an atrophic endometrium (< 4 mm) often struggle with infertility, menstrual irregularities, including amenorrhea, hypomenorrhea, and recurrent pregnancy losses. There is currently no specific treatment for these endometrial pathologies. Therefore, there remains a need to develop safe and effective therapies for the treatment of these pathologies.

[0004] NAGORI CHAITANYA B ET AL (JOURNAL OF HUMAN REPRODUCTIVE SCIENCES JAN 2011, vol.4, no.1, January 2011 (2011-01), pages 43-48) describes endometrial regeneration using autologous adult stem cells followed by conception by in vitro fertilization in a patient with severe Asherman syndrome.

[0005] GARGETT CAROLINE E ET AL (JOURNAL OF HUMAN-REPRODUCTIVE SCIENCES JAN 2011, vol.4, no.1, January 2011 (2011-01), pages 49-52) describes the generation of a receptive endometrium in Asherman syndrome.

[0006] FERYAL ALAWADHI ET AL (PLOS ONE, vol.9, no.5, 12 May 2014 (2014-05-12), page e96662) describes that bone marrow stem cell (BMDSC) transplantation improves fertility in a mouse model of Asherman's syndrome.

SUMMARY OF THE INVENTION

[0007] This invention relates, at least in part, to the discovery that autologous CD133+ bone marrow stem cells (BMDSCs) can regenerate vascularization leading to the generation of autologous functional endometrium de novo. Accordingly, the disclosure provides methods for initiating endometrial regeneration. The procedure may comprise administering an effective amount of autologous CD133+ bone marrow stem cells (BMDSC) into the uterine arteries of a subject in need thereof to initiate endometrial regeneration.

[0008] In one aspect the invention provides isolated autologous CD133+ bone marrow-derived stem cells (BMDSCs) for use in the treatment of Asherman's syndrome or endometrial atrophy in a subject in need thereof, wherein the isolated CD133+ BMDSCs are administered to the uterine arteries of the subject.

[0009] The subject is known to have Asherman syndrome or endometrial atrophy. In some embodiments, the subject has endometrial atrophy that is resistant to hormone treatment. In some embodiments, the subject has had one or more prior embryo implantation failures. In some embodiments, autologous CD133+ BMDSCs are prepared by administering to a subject an agent to mobilize BMDSCs from bone marrow into the subject's peripheral blood; and isolating CD133+ BMDSCs from the subject's peripheral blood. In some embodiments, the agent for mobilizing BMDSCs is granulocyte colony-stimulating factor (G-CSF). In some embodiments, autologous CD133+ BMDSCs are isolated from the subject's peripheral circulation via apheresis using an anti-CD 133 antibody. In some embodiments, CD133+ BMDSCs are administered to the uterine arteries via a catheter. In some embodiments, CD133+ BMDSCs are administered into the uterine spiral arterioles of a subject.

[0010] Some aspects of the disclosure provide a method for initiating endometrial regeneration, the method comprising isolating autologous CD133+ bone marrow stem cells (BMDSC) from a subject in need thereof; and administering an effective amount of the isolated CD133+ BMDSC into the uterine arteries of the subject to initiate endometrial regeneration.

[0011] In some embodiments, granulocyte colony-stimulating factor (G-CSF) is administered to the subject prior to isolation of autologous BMDSC.

[0012] The invention is defined in the patent claims. Each of the limitations of the invention may encompass various embodiments of the invention. Therefore, it is intended that any limitation of the invention that includes any element or combination of elements may be included in every aspect of the invention. The present invention is not limited in its application to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The invention may be in other embodiments and may be used or carried out in various ways. Also, the terminology and terminology used in this text are for purposes of description and should not be considered limiting. The use of "including," "comprising," or "has," "contains," "includes," and variations thereof herein, shall include the items listed below and their equivalents as well as additional items.

BRIEF DESCRIPTION OF THE PICTURES

[0013]

Sl. 1 is a schematic representation of the trial (A) and timeline (B) of the events shown in Fig. 1A. Sl. 2 is an angiogram showing the path of the probe from the uterine arteries through the spiral arterioles where CD133+ cells are located by non-invasive radiology.

Sl. 3 shows hysteroscopy of the uterine cavity from one patient with atrophic endometrium before, 3-6 and 9 months after autologous BMSC treatment.

Sl. 4 shows the thickness of the endometrium in 6 patients with atrophic endometrium/Asherman's syndrome included in this study, before and 3 months after autologous BMSC therapy.

Sl. Figure 5 shows the mean SD endometrial thickness before and 3 months after autologous BMSC treatment.

Sl. shows 3D ultrasound images showing the improvement in endometrial volume obtained 3 months after autologous BMSC therapy compared to the basal status before treatment.

Sl. 7A-7B show preoperative and postoperative hysteroscopic images. Hysteroscopic findings in patients with Asherman's syndrome (Fig. 7A) or atrophic endometrium (FIG. 7B) before stem cell therapy (1st examination), and 2-3 months (2nd examination) and 4-6 months (3rd examination) after stem cell therapy. The severity of endometrial adhesions was graded according to the American Fertility Society classification.

Sl. 8A-8I show tissue analyses. Immunohistochemical results for detection of mature blood vessels in endometrium from patient 7 before (FIG.8A), 3 months (FIG.8B) and 6 months (FIG.8C) after autologous cell therapy α-sma+, CD31+ positive cells identify mature blood vessels (20x). Fig. 8D shows human myometrium used as a positive control for α-sma staining, and human tonsil used as a positive control for CD31 (FIG. 8E). Sl.

8F shows a negative control resulting from the absence of primary antibody. Fig. 8G shows a detailed view of the vessel identified in Fig. 8C (40x). Figure 8H shows the dynamics of the total number of mature blood vessels in 8 patients before and 3 and 6 months after cell therapy, indicating a time-sensitive neoangiogenic effect. Fig. 8I shows statistical analysis of mean ± SEM of total mature blood vessels before and 3 and 6 months after treatment. Sl. 9 shows the trial design. Hysteroscopic reconfirmation and assessment of AS or EA was performed by one surgeon in the proliferative phase. BMDSC mobilization was induced by G-CSF injection, and five days later, CD133+ cells were isolated from peripheral blood through apheresis and immediately injected into spiral arteries by interventional radiology. The second and third hysteroscopies were apparently performed to evaluate the uterine cavity after stem cell treatment. Patients were then invited to try to conceive.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention is based, at least in part, on the discovery of a new therapeutic approach for initiating endometrial regeneration using autologous stem cell therapy. Specifically, the subject patent application is based on the finding that autologous CD133+ bone marrow stem cells (BMDSCs) can regenerate vascularization leading to the generation of autologous functional endometrium de novo. Although BMDSCs are known to be the source of non-hematopoietic cells in different endometrial cell compartments (stroma, glandular epithelium, and luminal epithelium), it is not known which subpopulation of BMDSCs promotes endometrial repair. The subject patent application provides safe and effective cell-based therapies for inducing endometrial regeneration and treating pathologies associated with endometrial degeneration such as Asherman's syndrome and atrophic endometrium.

[0015] The human uterus mainly consists of the endometrium, and the outer layer of smooth muscle is called the myometrium. The functional layer of the human endometrium is a highly regenerative tissue that undergoes monthly cycles of growth, differentiation and shedding during a woman's reproductive years. Fluctuating levels of circulating estrogen and progesterone dictate this dramatic remodeling of the human endometrium. Endometrial regeneration accompanies both childbirth and endometrial resection. Regeneration of the endometrium from the basal layer contributes to the replacement of the functional layer, followed by its detachment during menstruation and childbirth. However, the endometrium may not respond to estrogen and may not regenerate in certain pathologies, for example, Asherman's syndrome and atrophic endometrium. Such subjects may have abnormal endometrial proliferation and become infertile.

[0016] Asherman's syndrome (AS) (or Fritz's syndrome) is a condition characterized by adhesions and/or fibrosis of the endometrium most commonly associated with dilatation and curettage of the intrauterine cavity. A number of other terms have been used to describe the condition and related conditions, including: intrauterine adhesions (IUA), uterine/cervical atresia, traumatic uterine atrophy, sclerotic endometrium, endometrial sclerosis, and intrauterine synechiae. Trauma to the basal layer of the endometrium, for example, following a dilatation and curettage (D&C) performed after a miscarriage, or delivery, or for medical abortion, can lead to the development of intrauterine scars resulting in adhesions that can efface the uterine cavity to some extent. Ultimately, the entire cavity may be scarred and occluded. Even with a relatively small number of scars, the endometrium may not respond to estrogen, and the subject may have secondary menstrual irregularities (such as amenorrhea, hypomenorrhea, or oligomenorrhea) and become infertile. AS can also result from other pelvic surgeries, including cesarean sections, removal of fibroid tumors (myomectomy), and other causes such as IUDs, pelvic radiation, schistosomiasis, and genital tuberculosis. Chronic endometriosis of genital tuberculosis is a significant cause of severe intrauterine adhesions (IUA) in developing countries, often resulting in total obliteration of the uterine cavity that is difficult to treat.

[0017] Hysteroscopy is the gold standard for diagnosing AS. Imaging by sonohysterography or hysterosalpingography reveals the extent of scar formation. Depending on the severity, AS can lead to infertility, repeated miscarriages, pain from blood clots, and future obstetric complications. If left untreated, obstruction of menstrual flow resulting from adhesions can lead to endometriosis in some cases.

[0018] In atrophic endometrium, the endometrium becomes too thin due to low estrogen levels. To be considered atrophic, the thickness of the endometrium should be less than 4 - 5 mm on transvaginal ultrasound. The ratio of the size of the uterus to the cervix will also tend to decrease and may approach: 1. MRI may also show a decrease in endometrial thickness similar to that seen on ultrasound. Factors that can cause endometrial atrophy include prolonged oral contraception, a hypoestrogenic state (ovarian dysfunction), and the use of tamoxifen.

[0019] A procedure for reversing endometrial regeneration is described here. The procedure involves administering an effective amount of autologous CD133+ bone marrow stem cells (BMDSC) into the uterine arteries of a subject in need to initiate endometrial regeneration.

[0020] The method may comprise isolating autologous CD133+ bone marrow stem cells (BMDSC) from a subject in need thereof; and administering an effective amount of the isolated CD133+ BMDSC into the uterine arteries of the subject to initiate endometrial regeneration.

[0021] As used herein, "subject" includes all mammals, including, but not limited to, dogs, cats, horses, sheep, goats, cows, pigs, humans, and non-human primates. In some embodiments, the subject is female.

[0022] A subject in need of endometrial regeneration is a subject whose endometrium does not regenerate in response to estrogen and has a thin endometrial lining. Such subjects may commonly have abnormal endometrial proliferation and become infertile. The optimal thickness of the endometrial lining is between 10 and 15 mm, and the maximum thickness at the time of implantation is reached around the 21st day of a woman's menstrual cycle. In some embodiments, the subject in need of treatment has an endometrial thickness at the time of implantation that is less than 5 mm, less than 4 mm, less than 3 mm, less than 2 mm, or less than 1 mm. In some embodiments, the subject has menstrual irregularities characterized by decreased flow and duration of bleeding (amenorrhea, hypomenorrhea, or oligomenorrhea) and/or recurrent pregnancy losses.

[0023] The subject is known to have Asherman syndrome or endometrial atrophy. In some embodiments, the subject has endometrial atrophy that is resistant to hormone treatment. In some embodiments, the subject has had one or more prior embryo implantation failures.

[0024] It has been shown that bone marrow-derived stem cells (BMDSC) as an exogenous source contribute to tissue renewal and regeneration of various organs and tissues. In human and murine endometrium, BMDSCs are also a source of non-hematopoietic cells in different endometrial cell compartments (stroma, glandular epithelium and luminal epithelium). They mainly contribute to the formation of cells of the stromal endometrial chamber and, to a much lesser extent, the endometrial glandular and luminal epithelial parts.

[0025] BMDSCs include hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). However, which subpopulation of BMDSCs promotes endometrial repair is unknown.

[0026] The inventors of this patent application have demonstrated for the first time in humans the ability of bone marrow-derived CD133+ stem cells delivered to uterine arteries via surgical and catheter delivery systems to initiate endometrial regeneration. Autologous circulating CD133+ BMDSCs were isolated after previous bone marrow mobilization and re-implanted into uterine spiral arterioles of the same patient. CD133+ BMDSCs regenerate vascularization leading to the generation of autologous functional endometrium de novo. CD133 is a glycoprotein also known in humans and rodents as Prominin 1 (PROM1). It is a five-transmembrane cholesterol-binding protein that localizes to membrane protrusions and is often expressed on adult stem cells, where it is thought to function in maintaining stem cell properties by suppressing differentiation.

[0027] The CD133<+>BMDSC of the present invention may be derived from primary stem cells or may be derived from an established stem cell line. In some embodiments, the stem cells can be embryonic stem cells, adult stem cells, umbilical cord stem cells, somatic stem cells, bone marrow, or mobilized bone marrow stem cells. In preferred embodiments, the stem cells are adult stem cells.

[0028] In some embodiments, CD133+ BMDSCs are prepared by administering to a subject an agent to mobilize BMDSCs from the bone marrow into the subject's peripheral blood; isolation of CD133<+>BMDSCs from peripheral blood of subjects. In some embodiments, the stem cell mobilization agent is selected from the group consisting of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and plerixafor (AMD3100). In some embodiments, the agent for mobilizing the stem cells is G-CSF.

[0029] In some embodiments, autologous CD133<+>BMDSCs are isolated from the subject's peripheral circulation by a process called apheresis using anti-CD133 antibodies (see, e.g., Sovalat H, Scrofani M, Eidenschenk A, Pasquet S, Rimelen V, Hénon P. Identification and isolation from adult human bone marrow or G-CSF-mobilized peripheral blood CD34(+)/CD133(+)/CXCR4(+)/ Lin(-)CD45(-) cells, with morphological, molecular and phenotypic characteristics of very small embryonic (VSEL) stem cells.

2011 Apr;39(4):495-505). Apheresis, which is well known in the art, refers to a process or procedure in which blood is drawn from a donor subject and separated into its components, some of which are retained, such as stem cell populations, and the remainder returned to the donor by transfusion. Apheresis lasts longer than the entire blood donation. To collect blood, it takes about 10-20 minutes, while apheresis donation can take about 1-2 hours. Apheresis product refers to a heterogeneous population of cells collected from the apheresis process.

[0030] In some embodiments, CD133<+>BMDSC are isolated from isolated BMDSC using an anti-CD 133 antibody. In some embodiments, CD133<+>BMDSCs are selected using anti-CD133 antibodies until CD133<+>BMDSCs are at least 80%, 85%, 90%, 95%, 98%, 99%, 99.9%, or 100% pure. In some embodiments, the CD133<+>BMDSCs are at least 95%, 98%, 99%, 99.9%, or 100% pure.

[0031] Administration of CD133<+>BMDSCs, or therapeutic compositions containing such cells, to a subject in need may be accomplished, e.g., by transplantation, implantation (e.g., of the cells themselves or of the cells as part of a combination of matrix cells), injection (e.g., directly into uterine arteries), infusion, catheter delivery, or any other means known in the art to provide cellular therapy. In one embodiment, the cells are delivered by intra-arterial catheterization. The uterine artery catheterization procedure has been widely described and used in uterine myoma embolization (Ravina JH, Herbreteau D, Ciraru-Vigneron N, et al.

Arterial embolization for the treatment of uterine fibroids. Lancet 1995;346(8976):671 -2).

[0032] CD133<+>BMDSC can be administered in the uterine arteries of the subject. These arteries supply the uterus with blood. In some embodiments, CD133+ BMDSCs are administered into the uterine spiral arterioles of a subject. Spiral arteries are small arteries that temporarily supply blood to the endometrium of the uterus during the luteal phase of the menstrual cycle. These arteries are very sensitive to estrogens and progesterones, penetrate the functional layer of the endometrium, grow and send branches in it and show very different and unique patterns.

[0033] CD133<+>BMDSCs are administered in an effective amount. "Effective amount" refers to an amount sufficient to induce the desired biological response, ie. initiates the regeneration of the endometrium. An effective amount includes that amount required to slow, reduce, inhibit, alleviate, or reverse one or more symptoms associated with AS or endometrial atrophy. In some embodiments, these terms refer to:

Resumption of menses after CD133<+>BMSC stem cell treatment;

An increase in the thickness of the endometrium; (Endometrial thickness is measured as the length from the upper to the lower border of the myometrium in the fundus of the endometrial cavity. For example, the increase can be an increase of 50% of the maximum thickness ever obtained with hormone replacement therapy (HRT) as measured by the longitudinal axis of the ultrasound of the vagina at the bottom of the uterus (vgr of 4 to 6 mm);

Hysteroscopic and histological evidence of de novo endometrial formation; and/or

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The functionality of the reconstructed endometrium in terms of birth, pregnancy and implantation rates after embryo placement in these patients.

[0034] In some embodiments, at least 45 million CD133<+>BMDSCs are engrafted into the subject. In some embodiments, at least 50, 55, 60, 65 million CD133<+>BMDSCs are engrafted into the subject.

[0035] An effective amount can be determined by one skilled in the art using routine methods. In some embodiments, an effective amount is an amount that results in any improvement in the condition being treated. One skilled in the art can determine appropriate doses and ranges of therapeutic agents to use, for example, based on in vitro and/or in vivo testing and/or other knowledge of compound dosages. When administered to a subject, effective amounts of the therapeutic agent will, of course, depend on the particular disease being treated; severity of illness; individual patient parameters including age, physical condition, size and weight, concomitant treatment, frequency of treatment and route of administration. These factors are well known to one of ordinary skill in the art and can only be addressed with routine experimentation. In some embodiments, the maximum dose is used, i.e. the highest safe dose consistent with sound medical judgment.

[0036] The subject invention is further illustrated by the following Examples, which should in no way be construed as further limiting.

EXAMPLES

Example 1

Materials and methods

Design

[0037] The following is an experimental uncontrolled trial in 16 patients with refractory AS approved by the IRB at the Hospital Clinico de Valencia, Spain and funded by the Spanish Ministry of Health (Ref EC 11-299). BMDSC mobilization was performed using granulocyte-CSF (G-CSF) (5 mg/kg/12 h sc for 4 days). Seven days later, peripheral blood apheresis was performed with isolation of CD133+ cells. Then, autologous CD133+ cells were introduced into spiral arterioles by non-invasive radiological intervention through the uterine artery using a 2.5 F microcatheter. The status of the endometrial cavity was assessed by hysteroscopy, vaginal ultrasound and histology before and 3.6 and 9 months after the stem cell intervention.

Patients & methods

Inclusion criteria

[0038] Sixteen patients with a diagnosis of refractory Asherman's syndrome previously treated with surgery at least seven times or with endometrial atrophy (< 4 mm) resistant to hormonal treatment with recurrent failure were included in the trial. All patients were referred by their doctors worldwide to enter a clinical experimental trial supported by the Spanish Ministry of Health. The age of the patients was 20-45 years and all had normal liver, heart and kidney function. The absence of menstrual bleeding in the natural cycle or after hormone replacement therapy (HRT) was confirmed. Absence of psychiatric pathology, HIV, hepatitis B or C, and syphilis, as well as willingness to participate in the trial were also confirmed.

Exclusion criteria

[0039] Patients were excluded from the study if there was no peripheral venous access or if they had splenomegaly.

Methodology

1. Mobilization of bone marrow stem cells (BMSC)

[0040] In order to initiate the mobilization procedure, the following conditions are met:

The patient was informed about the procedure and received a consent form at least 24 hours before mobilization.

An appropriate medical assessment with relevant complementary investigations was carried out and confirmed by the recall physician from BMSC.

Relevant results of serological tests (HIV, HBcAg, HBsAg, HCV, syphilis) are available.

Veins were evaluated to determine their suitability for the procedure.

Then, BMSC mobilization into the peripheral blood was induced by G-CSF (5 mcg/kg sc every 12 hours) for 4 days.

2. BMSC re-collection

[0041] BMSC recollection was performed by a conventional apheresis procedure using a peripheral vein. Positive selection of CD133<+>cells was performed according to the PO-7610-02 protocol approved by the Hospital Clinico Universitario with the application of three washes and subsequent selection of CD133<+>cells. First, the cells were washed and incubated with a monoclonal antibody, then they were washed two more times, and finally subjected to CD133<+> selection.

[0042] The selection procedure was carried out for a maximum of 3 hours or until at least 50 million CD 133<+> cells were collected.

3. Transplantation of CD133<+> cells into uterine arterioles using intra-arterial catheterization

[0043] Twenty-four hours after their isolation, autologous CD133<+> cells were diluted in 15-30 cc of saline and then injected into spiral arteries. Cells were collected through a sterile syringe into a container and brought to the radiology department prior to their injection.

At least 45 million cells were implanted.

[0044] The uterine artery catheterization procedure has been widely described and used in the embolization of uterine fibroids. The required radiology equipment for this procedure was a radiosurgical C-arm or an angiography room with ultrasound examination. Briefly, after gaining access to the common femoral artery using the Seldinger technique, a 4F catheter was placed in the artery and used to catheterize both hypogastric arteries using a 2 cobra curve angiographic catheter and a 0.035 in. Terum guidewire. A 2.5 F microcatheter was placed with a 0.014 guidewire through the cobra catheter and the uterine artery was catheterized up to the ascending curve or until the microcatheter reached its most distal level. Once the catheter is stabilized and its position checked, CD133+ BMSCs are loaded into the saline suspension. The diameter of the cell injection catheter was 500-600 microns and 15cc was perfused.

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[0045] After the intervention, the patient stayed overnight in the hospital and was discharged the next day without complications.

Response criteria

[0046] This technique aims to repopulate the endometrial vascular niche in patients with Asherman's syndrome or endometrial atrophy using CD133<+>BMSCs, in order to reconstruct a functional endometrium that can allow embryo implantation in patients undergoing ART with recurrent endometrial failure. Therefore, the following indicators were taken into account for successful treatment:

Menstrual outcome, menses must restart after CD133<+>BMSC treatment. An increase in the thickness of the endometrium. A minimum of 50% of the maximum thickness ever obtained with HRT as measured by longitudinal axis of vaginal ultrasound at the bottom of the uterus (vgr of 4 to 6 mm) Hysteroscopic and histological evidence of de novo endometrial formation

The functionality of the reconstructed endometrium in terms of birth, pregnancy and implantation rates after embryo reimplantation in these patients.

Results

[0047]

Table 1. Clinical outcome after treatment with CD133+ stem cells

Table 2: Cycle length and amount and duration of menses in days after autologous CD133+BMDCC transplantation

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[0048] This is the first case series trial using this specific stem cell treatment administered intravascularly in AS. The frequency of AS varies between 2-22% of infertile women.

[0049] G-CSF is the most commonly used cytokine for BMSC mobilization in both autologous and allogeneic donors. This product is generally well tolerated. However, doses greater than 5 mcg/kg/day have been shown to result in musculoskeletal pain in more than 50% of cases. If this happens, paracetamol should be administered as an analgesic (500 mg/8 hours), while maintaining G-CSF. Other less observed complications are: nausea and vomiting, migraine and insomnia. In any case, symptomatic treatment should be applied. In general, symptoms disappear 3-4 days after stopping G-CSF, although asthenia may last up to 2 weeks after the last dose. Finally, splenic rupture in healthy donors has been associated with G-SCF administration. Therefore, an abdominal scan should be performed in all patients with pain in the left hypochondrium. Splenomegaly detected in these cases should be followed by immediate suspension of G-CSF. High levels of alkaline phosphatase and LDH are often detected without any associated symptoms. Leukocytosis is common, and values are usually less than 70 x10<9>/L.

Example 2

Study participants

[0050] Sixteen patients (range 30-45 years of age) diagnosed with either refractory Asherman syndrome (AS) based on the American Fertility Society classification (N=11) or endometrial atrophy (N=5) were invited to participate in the trial.

An earlier diagnosis of severe Asherman's syndrome or endometrial atrophy was confirmed, and hysteroscopies were performed in the proliferative phase. Patients diagnosed with AS were classified according to the AFS classification, and endometrial biopsies were obtained. All patients had little or no menstrual bleeding during natural cycles or after hormone replacement therapy (HRT). Requirements for participation in the trial included the following: normal liver, heart, and kidney function, absence of HIV, hepatitis B or C, syphilis, and psychiatric pathology, and willingness to complete the trial. Patients were excluded in cases where there was no peripheral vein access or splenomegaly.

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Mobilization and isolation of BMDSCs

[0051] Mobilization of BMDSCs was initiated by pharmacological administration of granulocyte colony stimulating factor (G-CSF) (10 µg/kg/day on days -4, -3, -2 and -1). G-CSF is a cytokine widely used for this purpose in autologous and allogeneic donors. Five days after injection, CD133+ cells were isolated by apheresis via peripheral veins using a CobeSpectra separator (Terumo BCT, Lakewood, CO). Two to three samples were processed per patient and a positive selection of CD133+ cells was obtained according to an established protocol using the CliniMACS® system (Miltenii Biotec GmbH, Bergisch Gladbach, Germany).

Selection was performed within three hours of collection until 50 million cells were obtained. Isolated CD133+ cells were diluted in 15 to 30 cc of saline and transported in a sterile syringe in the radiology department for delivery to spiral arterioles.

Delivery of BMDSCs

[0052] After successful CD133<+> isolation, patients were referred to the HCU radiology department, where intra-arterial catheterization was performed to deliver cells to the endometrial stem cell niche using a technique used for fibroid embolization. The common femoral artery was approached using the Seldinger technique, in which a 4F introducer allowed catheterization of both hypogastric arteries with an angiographic catheter curve and a Terumo guide (0.035 in). A 2.5 F microcatheter with a guidewire (0.014 in) was advanced through the latter catheter to catheterize the uterine artery to the longest spiral arterioles that the microcatheter could reach (Figure 9). Once the catheter position is stabilized and verified, 15 cc of saline suspension of selected CD133+ cells (containing 42 to 200 x10<6> cells, mean 123.56x10<6>± 57.64) is injected through each uterine artery into the spiral arterioles.

Continuation

[0053] All patients received hormone replacement therapy (Progiluton™, Bayer, Berlin, Germany) after receiving cell therapy. The status of the endometrial cavity was assessed by diagnostic hysteroscopy, vaginal ultrasound, and histology to determine endometrial thickness and the presence or absence of endometrial adhesions before, 2, 3, and 6 months after cell therapy. Patients were then invited to undergo ART to try

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conception (Figure 9).

Endometrial immunohistochemistry

[0054] Blood vessel formation was assessed by CD31 & α-sma-Cy3 immunohistochemistry in paraffin sections using anti-human CD31 (Dako, Glostrup, Denmark) with secondary Alexa goat anti-mouse 488 and mouse anti-human αsma-Cy3 (Sigma-Aldrich, MO, USA). Slides were counterstained with DAPI (Invitrogen, CA, USA). Positive controls included human tonsils for CD31 and myometrium for α-sma. The slides were examined under a fluorescent Nikon Eclipse 80i microscope. Three separate 20x fields were used to analyze total blood vessel formation per surface using ImageJ software. Data are presented as specific values for each patient before and 3 months and 6 months after cell therapy.

Statistical analysis

[0055] Statistical analysis was performed using SPSS 17.0 software (IBM, MD, USA). A paired sample t-test was used to analyze the difference in the number of total mature blood vessels. A P-value obtained in a 2-tailed test < 0.05 was considered statistically significant.

Results

[0056] Two patients were initially excluded from the trial due to poor mobilization of CD133+ cells (< 40 million) in one case and lack of peripheral venous access in the other. A total of 16 patients completed the protocol. No major complications were reported.

[0057] Patients were referred for investigation with a diagnosis of refractory AS (N=11) (Table 3). Patient menstrual histories revealed amenorrhea in two patients and minor spotting in nine. Causes of AS were traumatic dilatation and curettage (D&C) (N=9), hysteroscopic myomectomy (N=1), and unknown cause (N=1). The average number of previously attempted reparative operative hysteroscopies was two. No patient reported significant improvement in endometrial status despite surgical treatment. Three patients were classified as AS grade III, four patients were graded as EA grade II, two patients were classified as grade II, and one patient was classified as AS grade I (Figure 7A).

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The maximum endometrial thickness with high-dose HRT achieved before cell therapy was 4.3 mm ± 0.74 (range 2.7-5 mm) (Table 3).

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[0058] Patients with EA and implantation failure (N=5) (Table 4) included in this trial had prior menstrual amenorrhea (N=3) or scanty spotting (N=2).

Etiology was previous D&C (N=1), unexplained (N=1), levonorgestrel IUD use (N=1), premature ovarian failure (N=1), and previous hysteroscopic myomectomy (N=1). The average number of attempts at previous reparative operative hysteroscopies was two. Severe endometrial atrophy was observed in all cases (Figure 7B). The maximum endometrial thickness with high-dose HRT achieved before cell therapy was 4.2 mm ± 0.8 (range 2.75 mm) (Table 4).

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Endometrial reconstruction after stem cell therapy

[0059] After autologous CD133+ BMDSC therapy, menstrual cycles resumed with HRT in all 16 patients, except one with EA. However, the duration and intensity of menstruation, as assessed by the number of pads used, progressively decreased from a mean of 5.06 days (range, 3–7 days) in the first month to 2.12 (range, 1–3 days) in the sixth month after cell therapy (Supplemental Fig. 1A). Menstrual volume also decreased from a mean of 2.68 (range, 1-5) to 1.5 (range, 1-4) pads per day in the sixth month.

[0060] Uterine observations performed 2, 3 and 6 months after cell therapy revealed improvements in the endometrium and uterine cavity (Tables 3 and 4; Figure 7). Namely, all patients diagnosed with stage III improved to stage I, while one of the two patients diagnosed with stage II showed a completely normalized endometrial cavity, and the other improved to stage I. The remaining patient, initially diagnosed as stage I, improved from the qualifying score as shown in Table 3. The maximum postoperative endometrial thickness obtained was 6.7 mm (range 3.1-12 mm). (Table 3, Figure 7A). In the EA group, normal endometrium was observed after cell therapy in four of five patients (Table 4; Figure 7B). The maximum endometrial thickness obtained after cell therapy was 5.7 mm (range 5-12 mm) (Table 4).

[0061] The total number of mature blood vessels formed was assessed in 8 patients by colocalization of CD31 and α-sma performed before and 3 and 6 months after cell therapy (Figure 8). A gradual increase in blood vessel formation was noted after 3 months of treatment (patients 4, 5, 7, 12 and 13), while in others a constant number of mature blood vessels was found (patients 6, 9 and 10) (Figure 8H). To compare the results between the starting point of the experiment (referred to as control) and 3 months after the specific treatment with CD133+ cells, the respective means and SEMs of the data were examined. An increased number of total mature blood vessels (CD31+/α-sma+) was observed in patients after three months of treatment (p=0.021). These results indicate a characteristic neoangiogenesis after autologous injection of CD133+ cells in patients with AS and EA that progressively decreases after 6 months (Figure 8I).

[0062] The functionality of the reconstructed endometrium was evaluated according to the reproductive function

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the outcome of patients who wanted to become pregnant after autologous CD133+BMDSC therapy (Tables 3 and 4). Two patients conceived spontaneously, two or four months after cell therapy, resulting in pregnancy (patient 15) and miscarriage at 17 weeks due to premature rupture of membranes (patient 7). Six positive pregnancies were obtained after 13 embryo transfers, resulting in three biochemical pregnancies, one miscarriage at 9 weeks due to a chromosomally abnormal embryo after miscarriage, one ectopic pregnancy, and one ongoing pregnancy (patient 12). In one case, embryo transfer was canceled due to chromosomal abnormalities in all embryos (patient 8), and in another case transfer was not performed due to failure of cell therapy (patient 14).

Discussion

[0063] From a histological point of view, AS corresponds to the substitution of the endometrial stroma by fibrous tissue affecting the endometrial stem cells and, therefore, the function of the tissue. The glands are usually replaced by an inactive cuboidal epithelium, which generally does not respond to hormonal stimulation and causes a complete disappearance of the endometrial structure, encroaching on the endometrial stem cell niche and, therefore, tissue function. During the first 50 to 60 years after the discovery of AS, researchers focused on the prevalence, etiology, and pathology of the condition. With the advent of endoscopy, new methods were developed for the diagnosis and treatment of conditions; however, despite technological advances, about 50% of AS cases today have no comprehensive cure.

[0064] The first case of stem cell therapy specifically targeting the endometrial stem cell niche is described here. Under steady-state conditions, circulating EPCs (cEPs) represent only 0.01% of circulating cells. Therefore, mobilization of cEPs in combination with direct infusion in the affected organ is planned. Autologous CD133<+>BMDSCs were isolated after mobilization with G-CSF and then reintroduced into the spiral arterioles of the patient's uterus using noninvasive radiological procedures. CD133<+>BMDSCs regenerate vascularization and induce endometrial proliferation, leading to the generation of autologous reconstructed endometrium. CD133<+>BMDSCs have recently been investigated in regenerative medicine clinical trials in non-hematological applications.

[0065] The primary objective was endometrial reconstruction, assessed first by the restoration of menstruation, which occurred in 15 of 16 of our patients. Although the duration and intensity are

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periods gradually decreased six months after cell therapy, stem cell therapy had an immediate effect on endometrial morphology. Hysteroscopic visualization of the uterine cavity, endometrial thickness measured by vaginal ultrasound, and neoangiogenesis by immunohistochemistry were consistent with effective, albeit transient, endometrial reconstruction. A secondary objective was to test the functionality of the reconstructed endometrium by attempting conception. Several spontaneous pregnancies, with the use of ART, were achieved after cell therapy, and the two miscarriages recorded in this trial were not related to endometrial functionality.

[0066] Cell engraftment was a major concern, as the IRB would not allow labeling of CD133+BMDSCs with superparamagnetic iron oxide nanoparticles (SPIOs) to track the injected cells. Instead, an experimental immunodeficiency mouse model for Asherman's syndrome was used for this purpose. An aliquot of 1 million CD133+ BMDSCs from patients included in the study was used for further characterization and tested for Lgr5 cell and aldehyde dehydrogenase1 (ALDH1) activity, resulting in 75.72 ± 8% Lgr5 cells and 77.45 ± 7.81% ALDH1 activity, identifying stemness and progenitor cell status, respectively. Another aliquot of 1 million cells was incubated with 50 µg/mL Molday ION Rhodamine B for 18 h, resulting in a labeling efficiency greater than 97% in all experiments. Then, SPIO-labeled cells were injected into an immunodeficient mouse model of Asherman's syndrome through the tail vein or intrauterine injection. Cell engraftment was detected by identifying intracellular iron deposits using Prussian blue staining, revealing that CD133+ BMDSCs were embedded predominantly around endometrial vessels of traumatized endometrium.

[0067] A previous case report showed positive results in the treatment of AS with autologous isolation of CD9, CD40 and CD90 stem cells from the bone marrow and placing them in the endometrial cavity, while another report described the direct placement of uncharacterized mononuclear stem cells into the subendometrial zone with a needle. Both cases differ in the type of cells delivered and the targeted stem cell niche.

[0068] This trial demonstrates that CD133+ BMDSC autologous cell therapy is useful in the treatment of patients with refractory AS and EA who wish to conceive.

References

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[0070] Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

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Claims (9)

Patent claims 1. Isolated autologous CD133<+>bone marrow-derived stem cells (BMDSCs) for use in the treatment of Asherman's syndrome or endometrial atrophy in a subject in need thereof, wherein the isolated CD133<+>BMDSCs are administered into the uterine arteries of the subject. 2. Isolated CD133<+>BMDSC for use according to claim 1, wherein the subject has atrophic endometrium that is resistant to hormone treatment. 3. Isolated CD133<+>BMDSC for use according to claim 1 or according to claim 2, wherein the subject has one or more failed embryo implantations. 4. The isolated CD133<+>BMDSC for use according to any one of claims 1-3, wherein the isolated autologous CD133<+>BMDSC is isolated from the subject's peripheral blood after administration of the BMDSC mobilization agent to the subject from the bone marrow into the subject's peripheral blood. 5. Isolated CD133<+>BMDSC for use according to any one of claims 1-4, wherein the agent for mobilizing the BMDSC is granulocyte colony stimulating factor (G-CSF). 6. The isolated CD133<+>BMDSC for use according to any one of claims 1-5, wherein the CD133<+>BMDSC is administered into the uterine arteries via a catheter. 7. Isolated CD133<+>BMDSC for use according to any one of claims 1-6, characterized in that the CD133<+>BMDSC is administered to the spiral arterioles of the uterus of a subject. 8. Isolated CD133<+>BMDSC for use according to any one of claims 4-7, wherein the autologous CD133<+>BMDSC is isolated from the peripheral blood of the subject by apheresis and wherein the apheresis is performed using an anti-CD133 antibody. 9. Isolated CD133<+>BMDSC for use according to any one of claims 1-8, wherein at least 45 million CD133<+>BMDSC are administered to a subject.